Date of Graduation

12-2015

Document Type

Dissertation (PhD)

Program Affiliation

Biomedical Sciences

Degree Name

Doctor of Philosophy (PhD)

Advisor/Committee Chair

Dean A. Lee

Committee Member

Kimberly Schluns

Committee Member

Michele Redell

Committee Member

Joseph L. Alcorn Jr.

Committee Member

Shulin Li

Abstract

Natural Killer (NK) cells are cytotoxic lymphocytes, which play a critical role in the immune response against malignant cells and microbial infections. NK cells are equipped with activating receptors, which upon detecting ligands expressed on stressed cells induce cytolytic activity of NK cells. Stimulation of NK cell proliferation and priming of NK cytolytic capability are accomplished by cytokines, which mediate their signals mainly through JAK-STAT signaling pathway. Previously, we found that K562 cells genetically modified to express membrane bound IL-21 (mbIL-21), which predominantly activates STAT3, induce robust expansion and activation of human NK cells. Further investigations revealed role of STAT3 in the transcriptional regulation of NKG2D, a primary activating NK receptor. Based on these findings, I hypothesized that STAT3 signaling plays a critical role in human cytolytic function and proliferation.

I analyzed NK cells from Job syndrome patients to test my hypothesis. Job syndrome caused by dominant negative STAT3 mutations is a naturally occurring STAT3 deficient genetic model. Assessment of cytolytic activity revealed impaired cytolytic function in Job Syndrome patients’ NK cells. Investigations into the probable underlying causes of impaired cytotoxicity showed deficient NKG2D receptor expression and impaired polarization of cytolytic granules to the immune synapse formed between Job syndrome patients’ NK cell and target cell. I validated these findings in STAT3 knock-down primary human NK cells, which also displayed impaired cytolytic function and cytolytic granule polarization. Expansion of Job syndrome patients’ NK cells with mbIL21 stimulation restored NKG2D expression and cytolytic granule polarization to normal levels and enhanced cytolytic activity. As constitutively active STAT3 is oncogenic, STAT3 is major drug target in cancer therapeutics. To assess a probable side effect of pharmacological inhibition of STAT3, I assessed its effect on human NK cell cytolytic function. Treatment of primary human NK cells with small molecule STAT3 inhibitor S3I-201 suppressed NK cytolytic function.

I employed pharmacological and genetic models of STAT3 deficiency to study the role of STAT3 in human NK cell proliferation. Treatment with STAT3 inhibitor S3I-201 reduced expansion of human NK cells stimulated with mbIL21 and membrane bound IL-15 (mbIL15). mbIL21 and mbIL15 induced expansion was also deficient in Job syndrome patients’ NK cells. Thus, both pharmacological and genetic models complemented each other in underlying role of STAT3 signaling in human NK cell proliferation.

Employing pharmacological and genetic approaches, I showed that STAT3 deficiency in primary human NK cells causes impairment of cytolytic function and cytokine induced expansion. This is the first report to demonstrate the role of STAT3 in the transport of cytolytic granules in NK cell and NK cell functional deficiency in Job syndrome patients, which may provide an immunological basis for their proclivity to cancer. Restoration of NK cell function upon mbIL21 stimulation, suggests adoptive NK cell therapy as a treatment option for Job syndrome patients. By assessing the effect of pharmacological STAT3 inhibition on NK cytotoxicity and proliferation, this study provides potential biomarkers for monitoring side effects of STAT3 inhibition, which is fast emerging as a therapeutic approach in cancer treatment.

Keywords

NK, STAT3, Job syndrome, Cytolytic function, MTOC polarization, S3I-201, NKG2D